Neurotransmitter release is acutely triggered by Ca2+ and is regulated in presynaptic plasticity processes that may underlie learning and memory. Characterization of the mechanisms of release and its regulation is thus critical to understand brain function and will facilitate the development of therapies for neurological diseases with a presynaptic origin. Several proteins with critical roles in release contain multiple C2-domains, which are widespread Ca2+-dependent phospholipid-binding modules that also exhibit Ca2+-independent activities. These proteins include: i) synaptotagmin 1, the Ca2+ sensor that triggers fast release; ii) other synaptotagmin isoforms, which likely act as alternate Ca2+ sensors and/or regulate the Ca2+ sensitivity of release; iii) munc13-1, which is essential for synaptic vesicle priming and mediates augmentation of release by phorbol esters; and iv) RIM1, a Rab effector that is also involved in vesicle priming and is essential for mossy fiber long-term potentiation (LTP). The C2-domains of these proteins are highly conserved and likely regulate neurotransmitter release at multiple levels through their Ca2+-dependent and Ca2+-independent interactions. To gain insight into these diverse functions of C2-domains in release, we propose an integrated approach involving structural, biochemical, genetic and electrophysiological experiments. We will study in detail the interactions of the synaptotagmin 1 C2-domains with phospholipids and with components of the membrane fusion machinery known as SNAREs, and will correlate our results with electrophysiological analyses of synaptotagmin 1 function in vivo to shed light on how it triggers release. We will also compare the Ca2+, phospholipid and SNARE-binding properties of the synaptotagmin 1 and 2 C2-domains, and will test the hypothesis that synaptotagmin 2 acts as an alternate Ca2+-sensor in triggering fats release. We will also analyze these properties in the C2-domains from other synaptotagmin isoforms and will perform mutagenesis experiments to gain insight into the biophysical basis for differences that may underlie distinct types of Ca2+ regulation of release. To help understand the functions of the RIM1 and munc13-1 C2-domains, which are mostly Ca2+-independent, we will determine their three-dimensional structures and will analyze their interactions with diverse target molecules, with particular emphasis on interactions that likely underlie plasticity processes such as mossy fiber LTP and phorbol-ester dependent augmentation of release.